Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

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DOI: 10.1371/journal.pone.0066654 · Source: PubMed
Abstract
Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.
Coordinated Binding of Single-Stranded and Double-
Stranded DNA by UvsX Recombinase
Robyn L. Maher, Scott W. Morrical*
Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont, United States of America
Abstract
Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork
restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the
RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are
activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination,
UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA
in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected
by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two
DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in
the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the
enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an
inactive form prior to ssDNA generation.
Citation: Maher RL, Morrical SW (2013) Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase. PLoS ONE 8(6): e66654.
doi:10.1371/journal.pone.0066654
Editor: Sergey Korolev, Saint Louis University, United States of America
Received October 27, 2012; Accepted May 11, 2013; Published June 18, 2013
Copyright: ß 2013 Maher, Morrical. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health research grant no. R01GM48847 to SWM and by American Cancer Society postdoctoral
fellowship no. PF-09-254-01-DMC to RLM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: smorrica@uvm.edu
Introduction
Unrepaired DNA double-strand breaks are associated with
increased risk of certain cancers in humans [1,2]. Homologous
recombination (HR) is used by cells for error-free repair of DNA
double-strand breaks, and to restart stalled replication forks [3,4].
Recombinases of the RecA/Rad51 family perform the central
catalytic role in homologous recombination of DNA. Recombi-
nases bind cooperatively to regions of single-stranded DNA
(ssDNA) to form a presynaptic filament. The presynaptic filament
is then aligned with homologous regions of double-stranded DNA
(dsDNA), and a strand exchange occurs in which the single-
stranded DNA invades and disrupts the double-stranded DNA to
exchange out one of the strands. The recombinase filament then
must dissociate from the DNA to allow for completion of the
repair process [5,6].
Presynaptic filament formation on ssDNA is necessary to
activate the recombination activities of UvsX and other recombi-
nases. Recombinases must also negotiate interactions with dsDNA
during at least three stages of the strand exchange process: First,
presynaptic filament assembly on ssDNA must occur in the
presence of a large excess of dsDNA. Therefore recombinase
filaments that form inappropriately on dsDNA must be resolved
and the recombinase subunits redirected into productive filament
assembly on ssDNA. Second, once presynaptic filaments assemble,
they must recognize homologous dsDNA as a substrate. Third,
following strand invasion, the recombinase filament bound to the
double-stranded, heteroduplex product must dissociate to provide
access to downstream DNA replication and repair machineries.
Thus, recombinase-dsDNA interactions occur during presynapsis,
synapsis, and postsynapsis stages of DNA strand exchange. Proper
coordination between recombinase-ssDNA and dsDNA-binding
activities must occur to ensure that recombination happens in a
timely and efficient manner.
To effectively catalyze the strand exchange reaction, recombi-
nases work in concert with several other proteins. These include
single-stranded DNA binding proteins (SSBs) that denature
secondary structure in single-stranded DNA and regulate access
to ssDNA during various stages of DNA metabolism [7]. Most
recombinases cannot compete with SSBs to gain access to ssDNA.
As a result, recombinases require another family of proteins called
recombination mediator proteins [8]. These proteins facilitate the
displacement of SSBs and regulate the activity of recombinases
such that recombination does not occur at inappropriate times or
places in the genome. Evidence suggests that recombination
mediator proteins also play important roles in directing recombi-
nase assembly onto ssDNA during presynapsis [6,9,10]. However
recombinases themselves must have some intrinsic properties that
allow them to identify appropriate DNA substrates throughout the
strand exchange process. It is these properties that are the subject
of this study.
The general mechanisms of recombination described above are
conserved throughout most DNA based organisms. The bacterio-
phage T4 recombination system is one of the most simple and
robust [11,12]. In comparison to the prokaryotic and eukaryotic
systems, the T4 recombination system has relatively few regulatory
and accessory factors. This makes UvsX ideal for detailed studies
of the fundamental mechanism of recombinase-catalyzed DNA
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66654
strand exchange, which is likely to be conserved in higher
organisms. UvsX is orthologous to the bacterial RecA, the
eukaryotic Rad51 and the archaeal RadA recombinase families
[13–15].
The ssDNA binding properties of UvsX protein have been
studied under a variety of conditions [16,17]. UvsX binds
cooperatively to long ssDNA molecules with a binding site size
of 4 nucleotides per UvsX monomer. In the presence of ATP,
DNA within the UvsX filament is stretched and underwound,
disrupting base stacking interactions [18,19]. UvsX/ssDNA
filaments are stabilized in the stretched/underwound form by
adenosine-59-(3-thio)-triphosphate (ATPcS), a slowly hydrolyzed
ATP analogue [16,20]. Much less is known about the dsDNA
binding properties of UvsX protein. It is known that UvsX binds
to dsDNA with affinity that is at least as strong as its affinity for
ssDNA [21] (H. Xu and S. Morrical, unpublished results). Unlike
ssDNA, dsDNA binding does not activate the ATPase activity of
UvsX [22]. Quantitative details of UvsX-dsDNA binding, and its
relationship to nucleotide and ssDNA binding, are generally
lacking, however.
To understand the mechanism by which UvsX discriminates
between ssDNA and dsDNA, it is necessary to devise methods for
measuring its affinity for one in the presence of the other. In this
study we quantitatively characterize and directly compare UvsX
interactions with short, fluorescently tagged homopolymeric
ssDNA and dsDNA substrates. The use of these substrates
eliminates secondary structure and long-range cooperative effects,
revealing the fundamental allosteric effects caused by one DNA
ligand on the binding of the other. We find here, as in other studies
[21], that UvsX binds to dsDNA alone better than to ssDNA
alone. We show that the two DNA binding sites of UvsX are
regulated by the nucleotide cofactor ATP and are coordinated
with each other such that in the presence of ssDNA, dsDNA
binding affinity is reduced and correlated with its homology to the
bound ssDNA. The data suggest a mechanism in which ssDNA
binding allosterically reduces UvsX-dsDNA binding affinity,
allowing UvsX to sample dsDNA for homology without the
overabundance of heterologous dsDNA in the cell becoming
inhibitory.
Materials and Methods
DNA Oligonucleotides and Nucleotide Cofactors
Oligonucleotide 1 (oligo 1) is a 25 mer of the sequence 59-
dA
22
XA
2
-39 in which X is amino-modifier C2 dT (Glenn
Research) and was synthesized and purified by HPLC by
Biosynthesis Inc., Lewisville TX. The amino linker was reacted
with AlexaFluor 546 carboxylic acid succinimidyl ester (Invitro-
gen, Eugene, OR) according to the supplier’s protocol to generate
the labeled substrate. Spectral analysis indicated that the
oligonucleotide was .95% labeled and free of non-covalently
associated fluorophore. Oligonucleotide 2 (oilgo 2) is a 25 mer of
the sequence dT
25
. Oligonucleotide 3 (oligo 3) is a 25 mer of the
sequence dC
25
. Oligonucleotide 4 (oligo 4) is a 25 mer of the
sequence dA
25
. Oligonucleotides 2–4 were synthesized and
purified by HPLC (IDT, Coralville IA). Double-stranded DNA
substrates were generated by mixing equimolar amounts of the
complementary oligonucleotides, heating the mixture to 80uC, and
allowing for slow cooling to room temperature for approximately
16 hours. Homopolymeric double-stranded oligos were analyzed
by non-denaturing polyacrylamide gel electrophoresis (PAGE) and
found to migrate exclusively as double-stranded 25 mers. All
concentrations of DNA are stated in nucleotides (ssDNA) or base
pairs (dsDNA). ATP was purchased as an HPLC purified solution
(pH 7.5) (GE Healthcare). ATPcS (Sigma) was purchased in
powdered form and resuspended in Tris base to generate a pH
neutral solution.
Oligonucleotide Strand Exchange Assays
59-[
32
P]-labeled dsDNA was generated by incubating oligo 4
with T4 polynucleotide kinase (Invitrogen) and c-[
32
P]-ATP for
1 hour at 37uC. The kinase was heat inactivated at 70uC for
10 min. Equimolar amounts of [
32
P]-labeled oligo 4 and unlabeled
oligo 2 were added together in reaction buffer (20 mM Tris-HCl,
pH 7.4, 50 mM NaCl, 3 mM MgCl
2
), heated to 80uC and
allowed to anneal through slow cooling to room temperature.
Strand exchange reactions were initiated upon addition of UvsX
(2 mM) to 8 mM (nucleotides) single-stranded dA
25
(oligo 4) and
2 mM double-stranded dA
25
:dT
25
(oligo 4: oligo 2), and 2.5 mM
ATP in reaction buffer. Reactions were conducted at room
temperature. Aliquots were removed from the reaction and
quenched at various time points in 50 mM EDTA, 1% SDS,
1X loading dye (Invitrogen). Samples were analysed using a 1X
TBE 4–20% gradient native acrylamide gel (BioRad). The gel was
dried under vacuum at 70uC for 45 min. Gels were visualized
using the Bio-Rad Molecular Imager FX phosphorimaging system
provided by the Vermont Cancer Center.
UvsX Protein
UvsX protein was purified using a modification of previously
published methods [22]. DNA encoding the UvsX protein was
propagated in a pET27b expression vector (Novagen) and used to
recombinantly overexpress UvsX protein in BL21 (DE3) E.coli cells
(Stratagene). Cells were lysed via sonication and the soluble
portion was obtained after centrifugation. This lysate was applied
to a DEAE cellulose (Whatman) ion exchange column (column
buffer 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM EDTA,
10% glycerol, 5 mM BME). Proteins were fractionated during
elution from the column with a gradient application of column
buffer containing 500 mM NaCl. Fractions containing UvsX were
identified by SDS-PAGE analysis and applied to a hydroxylapatite
column (Bio-Rad Laboratories) (column buffer 10 mM K
2
HPO
4
pH 7.4, 100 mM NaCl, 10%glycerol, 5 mM BME). Proteins were
fractionated during elution from the column with a gradient
application of column buffer containing 700 mM K
2
HPO
4
pH 7.4. The protein elutes from this column in both ‘‘early’’
and ‘‘late’’ eluting fractions. The ‘‘early’’ eluting fraction has no
DNA binding or strand exchange activity. The late eluting fraction
has these activities and was relatively homogeneous. This fraction
was loaded onto a HiTrap Q HP (GE Healthcare) ion exchange
column (column buffer 20 mM Tris-HCl, pH 7.4, 75 mM NaCl,
5 mM EDTA, 10% glycerol, 5 mM BME). Proteins were
fractionated during elution from the column with a gradient
application of column buffer containing 500 mM NaCl. Protein
purity was analyzed by SDS-PAGE with Gel Code Blue (Thermo)
protein stain. The protein was found to be greater then 95% pure,
and was nuclease-free according to published criteria [23]. Protein
was concentrated as necessary using Vivaspin centripetal concen-
trators (Sartorius Stedium), and stored at 280uC in storage buffer
(20 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.2 mM EDTA, 10%
glycerol, 1 mM DTT). Protein concentrations were determined by
absorption at 280 nm using an extinction coefficient of
69,760 M
21
cm
21
.
Steady-state Fluorescence Measurements
Steady-state fluorescence measurements were made using a
Quantamaster-6 fluorometer (PTI, Birmingham NJ). The excita-
tion monochromator was set to 554 nm with 1 nm band pass slits.
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 2 June 2013 | Volume 8 | Issue 6 | e66654
Spectra were taken from 560–600 nm with emission monochro-
mator band pass slits set at 5 nm. Binding reactions contained
2 mM ssDNA (AlexaFluor 546-labeled oligo 1) or dsDNA
(AlexaFluor 546-labeled oligo 1:oligo 2), 2 mM UvsX and
2.5 mM ATP in reaction buffer (20 mM Tris-HCl, pH 7.4,
50 mM NaCl, 3 mM MgCl
2
) at a total final volume of 80 mlat
25uC.
Stopped-flow Fluorescence Measurements
Stopped-flow fluorescence measurements were made using an
SX.18 MV stopped-flow fluorometer (Applied Photophysics,
Leatherhead, Surrey, UK). Excitation monochromator was set at
550 nm with slits of 9.3 nm and a 570 nm long-pass filter was
placed in front of the detector with slits of 9.3 nm. Protein, DNA,
and nucleotide reagents in 20 mM Tris-HCl, pH 7.4, 50 mM
NaCl, 3 mM MgCl
2
reaction buffer, at 25uC, were mixed from 2
syringes according to the schematic provided with each figure.
The fluorescently labeled species is indicated with an asterisk.
Rapid mixing was in a 1:1 ratio. At least 2 shots were averaged for
each reaction and at least 3 independent reactions were averaged
to generate the kinetic parameters. All binding reactions were
monitored for at least 30 s. Just after mixing a decrease in
fluorescence was observed indicative of binding, and after several
seconds a lower resting level of fluorescence was observed
indicating that equilibrium had been achieved (Figure S1).
Reaction progress curves were fit to an exponential function
(Graphpad) to determine the amplitude of fluorescence quenching
at equilibrium, I
b
=(|F
0
-F
b
|), in which F
0
is the fluorescence
intensity of the free DNA and F
b
is the fluorescence intensity of the
DNA following addition of protein (both in arbitraty units). In
reactions containing ATP and ssDNA, ATP hydrolysis catalyzed
by UvsX eventually destabilizes UvsX-ssDNA interactions, result-
ing in loss of fluorescence quenching after a time delay. At earlier
time points, however, these traces parallel those obtained in the
presence of ATPcS (Figure S1). Therefore in these experiments
the amplitude of fluorescence quenching was determined during
the transient equilibrium period prior to the onset of signal
recovery.
Amplitudes of fluorescence quenching were graphed as a
function of UvsX concentration and fit to a quadratic binding
equation (Equation 1) to determine an apparent equilibrium
dissociation constant (K
d
) [24,25] in which, E
t
is the total enzyme
present, B
t
is the total DNA binding sites present, I
f
is the
amplitude of fluorescence quenching of the free DNA (always
floats close to zero), I
b
is the amplitude of fluorescence quenching
of the bound DNA upon binding, I
obs
is the observed amplitude of
fluorescence quenching at any given protein concentration.
Equilibria were achieved within 30 seconds of mixing for all
reactions for which apparent K
d
data are listed. This analysis
assumes a 4 bp/UvsX binding site size. This binding site size has
been measured for ssDNA in several other studies [16,22] and for
short dsDNA in this study (Figure S5).
Iobs~
(KdzEtzBt){
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
((KdzEtzBt)
2
{4EtBt)
q
2Bt
0
@
1
A
(Ib{If )zIf
ð1Þ
Results
Monitoring UvsX-DNA Interactions by the Quenching of
AlexaFluor 546 Fluorescence
Previous studies established that the quenching of a DNA-
bound fluorophore provides a reliable, quantitative read-out on
the stability of UvsX-DNA interactions [20]. Here, oligonucleotide
1, dA
22
XA
2
, was labeled with AlexaFluor 546 as a probe to detect
UvsX binding to DNA. A dsDNA substrate was generated by
annealing the complimentary oligo 2 to the AlexaFluor 546
labeled oligo 1. Annealing of the complementary oligonucleotide
did not change the fluorescence intensity of the labeled oligo 1
(free ssDNA and free dsDNA have the same fluorescence
intensity). Non-denaturing PAGE analysis of the annealed product
verified that it migrates exclusively as a duplex 25 mer (data not
shown). Figure 1A shows the fluorescence emission spectrum of the
free dsDNA substrate labeled with AlexaFluor 546 (2 mM
nucleotide pairs) (solid line). The addition of UvsX (2 mM)
generated a ,25% quench in probe fluorescence with no spectral
shift (dashed line). The addition of ATP (2.5 mM) did not affect
the level of quenching appreciably, but the spectrum is slightly red-
shifted (dotted line).
Similar methods were used to detect UvsX binding to ssDNA.
Figure 1B shows the fluorescence emission spectra of labeled oligo
1 alone (solid line) (2 mM nucleotides). Addition of UvsX (2 mM)
did not change the intensity of the emission (dashed line). However
the addition of ATP (2.5 mM) caused a ,25% decrease in the
intensity of the fluorescence emission and a slight red-shift in the
Figure 1. Effects of UvsX protein on fluorescence emission
spectra of AlexaFluor 546-labeled oligonucleotides. Conditions
were as described in Materials and Methods. The fluorescence emission
spectra of (A) 2 mM (nucleotide pairs) double-stranded oligonucleotide
dT
25
:dA
22
XA
2
(oligo 2:oligo 1), and (B) 2 mM (nucleotides) single-
stranded oligonucleotide dA
22
XA
2
(oligo 1), were recorded in the
absence of UvsX (solid line), in the presence of a saturating amount of
UvsX (2 mM) (dashed line), and in the presence of saturating amounts of
both UvsX and ATP (2.5 mM) (dotted line).
doi:10.1371/journal.pone.0066654.g001
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 3 June 2013 | Volume 8 | Issue 6 | e66654
spectra indicative of UvsX binding (dotted line). Similar results
were obtained when the position of the AlexaFluor 546 probe was
moved to the 59 end of the oligo (Figure S2). The data therefore
indicate that stable binding to single-stranded oligo 1 is ATP-
dependent under the conditions of our experiments. We have
found that, like other recombinases, the binding of UvsX to
ssDNA is influenced by the base composition of the polynucleotide
[26]. Oligo 1 is essentially a dA homopolymer. UvsX binding to
AlexaFluor 546-labeled oligo dT can be detected in the absence of
ATP, however (Figure S3). This differential dependence on ATP is
most likely due to differences in base stacking interactions in these
sequences (see Discussion).
There was some concern that the presence of the AlexaFluor
546 label might alter the affinity of UvsX for labeled versus
unlabeled DNA. Therefore we conducted experiments similar to
those in Figure 1 using different ratios of labeled/unlabeled DNA.
Results of these competition experiments indicate that the
presence of the AlexaFluor 546 label on DNA does not
significantly alter the DNA-binding affinity of UvsX (data not
shown). Subsequent experiments make use of the fluorescence
changes described in Figure 1 for quantitative analysis of UvsX-
DNA interactions.
UvsX Binding to dsDNA in the Presence of ATP and
ATPcS
To allow for accurate comparisons with ssDNA binding data,
fast mixing techniques were used to quantify UvsX-dsDNA
interactions, even though dsDNA does not activate ATP
hydrolysis by UvsX [22]. To measure binding affinity for the
dsDNA substrate, various amounts of UvsX were incubated in one
syringe and then rapidly mixed with 0.5 mM (nucleotides, final)
AlexaFluor 546-labeled dsDNA. The amplitude of fluorescence
quenching was graphed as a function of UvsX concentration
(Figure 2A). These data were fit to Equation 1 to determine an
apparent dissociation constant of 46618 nM (Table 1). This
analysis assumes a dsDNA binding site size of 4 bp, which was
verified by stoichiometric titration under tight binding conditions
(Figure S5), and which is equivalent to the ssDNA binding site size
of 4 nucleotide residues [16,17]. The use of the quadratic binding
model in Equation 1 is justified by the observation that UvsX
binding to short DNA molecules is not cooperative, as shown by
the non-sigmoidal binding data in Figures 2–3. This feature of
UvsX-DNA interactions is explored further in the Discussion.
In Figure 1A, ATP was not required for UvsX binding to
dsDNA, however a slight red shift was observed in the presence of
ATP. This may indicate that ATP is bound under these conditions
and that it may affect the binding affinity or conformation of the
UvsX filament on dsDNA. Similar assays as those described above
were performed to measure the binding affinity for UvsX to
dsDNA in the presence of ATP or ATPcS. Absolutely higher
concentrations of both UvsX and dsDNA were required to
measure accurately this elevated apparent K
d
. The concentrations
of ATP and ATPcS used in these experiments were saturating as
determined in a separate assay (Figure S4). To determine the
binding affinities various amounts of UvsX were rapidly mixed
Table 1. Apparent dissociation constants for UvsX binding to
dsDNA or ssDNA
a
.
Other Ligands Present
Apparent
K
d
(nM)
dsDNA ssDNA
None 46618 Not bound
b
ATP 170660 7606140
ATPcS230650 740690
ATP+homologous ssDNA 10506300
ATP+heterologous ssDNA N.D.
c
ATP+homologous dsDNA #750
d
a
Equilibrium binding data derived from Figures 2, 3, 4, and 6.
b
No binding observed under the experimental conditions used.
c
Not Determined. Measuring the apparent K
d
for dsDNA in the presence of ATP
and heterolog ous ssDNA requires unattainably high protein concentrations.
d
Based on observation in Figure 4 that homologous dsDNA does not
destabilize UvsX-ssDNA interactions.
doi:10.1371/journal.pone.0066654.t001
Figure 2. Binding of UvsX to double-stranded DNA in the
absence or presence of nucleotide cofactors. Reactions were
initiated by the addition of UvsX (final concentrations indicated on
graph) to dsDNA, oligonucleotide dT
25
:dA
22
XA
2
(oligo 2:oligo 1) and
nucleotide cofactors final concentrations are indicated below. The
amplitude of fluorescence quenching was graphed as a function of
UvsX concentrations and fit to Equation 1 to determine an apparent K
d
value. (A) 0.5 mM (nucleotide pairs) dsDNA (B) 2 mM dsDNA and 2.5 mM
ATP (C) 2 mM dsDNA and 900 mM ATPcS.
doi:10.1371/journal.pone.0066654.g002
Coordinated ss/dsDNA Binding by UvsX Recombinase
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with 2 m M (final, base pairs) of the labeled duplex and either ATP
(2.5 mM) or ATPcS (900 mM). The amplitude of fluorescence
quenching was graphed as a function of protein concentration
(Figure 2B–C). These data were fit to Equation 1 to determine
apparent K
d
values of 170660 nM (ATP) and 230650 nM
(ATPcS), respectively. Based on these values, it is evident that
UvsX binds to dsDNA with 4 to 5-fold reduced affinity in the
presence of ATP or ATPcS (Table 1).
UvsX Binding to ssDNA in the Presence of ATP and ATPcS
Similar rapid mixing assays were performed to determine the
binding affinities of UvsX to ssDNA in the presence of substrate
ATP or substrate analog ATPcS. A previous study established that
UvsX hydrolyzes ATPcS very slowly in the presence of ssDNA
[27]. The amounts of nucleotide cofactor used were saturating as
determine in a separate assay (Figure S4). Various amounts of
UvsX were incubated in one syringe and then rapidly mixed with
2 mM (nucleotides, final) labeled oligo 1 and 900 mM ATPcS
(final) or 2.5 mM ATP (final). The amplitude of fluorescence
quenching was graphed as a function of UvsX concentration
(Figure 3A–B). These data were fit to Equation 1 to determine
apparent K
d
values of 740690 nM in the presence of ATPcS and
7606140 nM in the presence of ATP (Table 1). The similarity of
these two dissociation constants in the presence of these two
nucleotide cofactors indicates that our methods have most likely
captured the dissociation constant of the ATP bound species
before hydrolysis. Consistent with this, results of coupled ATPase
assays indicate that ADP release is not detectable on the time scale
of the reactions in Figure 3 (#30 s) (data not shown). These
observations are important because the ATP bound species is
transient but it is also thought to be the form of the enzyme that is
critical for homology search [28,29]. This will be a key factor in
later experiments when homologous and heterologous substrate
pairs are present simultaneously.
UvsX Binding to ssDNA in the Presence of ATP is
Unaffected by Homologous dsDNA
The apparent K
d
values measured for ssDNA in the presence of
ATP/ATPcS are 3–4 fold greater than those measured for
dsDNA under identical conditions (Table 1). This may be
counterintuitive since it is thought that the recombinase filament
must form on ssDNA to initiate strand exchange properly.
However these apparent K
d
values were only determined in the
presence of one polynucleotide substrate or the other. To more
closely mimic DNA strand exchange conditions, we measured the
affinity of UvsX for ssDNA and dsDNA substrates when they are
present simultaneously. The reaction can be monitored from the
perspective of each oligonucleotide, by placing the AlexaFluor 546
probe on either the dsDNA or the ssDNA. Then how the presence
of one substrate, at one site on the enzyme, may affect binding of
the other substrate at another site on the enzyme can be
determined. All reactions were conducted in the presence of a
saturating amount of ATP.
AlexaFluor 546-labeled ssDNA and unlabeled dsDNA were
used to measure ssDNA binding in the presence of increasing
dsDNA concentrations. The binding of UvsX to labeled ssDNA
was monitored under conditions in which the ssDNA is saturated
with UvsX (see Figure 3A) and unlabeled dsDNA of homologous
sequence is titrated in. UvsX was simultaneously mixed with both
DNA substrates and a saturating amount of ATP. Figure 4 shows
3 mM UvsX (final) binding to 2 mM labeled ssDNA (final,
nucleotides) in the presence of 2.5 mM ATP and increasing
amounts of homologous dsDNA. Reaction progress curves were fit
to an exponential function to determine the amplitude of
fluorescence quenching. The amplitude was graphed as a function
of homologous dsDNA concentration and data were fit to a line to
demonstrate the trend (solid line). There is no change in the
amplitude of fluorescence quenching in the presence of ever
increasing amounts of homologous dsDNA, indicating that ssDNA
binding is not destabilized in the presence of dsDNA. These results
suggest that the ssDNA and dsDNA substrates do not directly
compete for the same binding site on UvsX. However the
possibility still exists that ssDNA binding is not destabilized in the
presence of homologous dsDNA because the dsDNA, which is not
labeled in this experiment, is not bound. This possibility is
eliminated in subsequent experiments.
UvsX Binding to dsDNA is Destabilized in the Presence of
ATP and ssDNA of Homologous Sequence
Similar reactions as those above were conducted to determine if
dsDNA binding is changed in the presence of ssDNA. Labeled
dsDNA and unlabeled ssDNA were used to measure dsDNA
binding in the presence of increasing ssDNA concentrations. The
binding of UvsX to AlexaFluor 546-labeled dsDNA was moni-
tored under conditions in which the dsDNA is saturated with
UvsX (see Figure 2B) and unlabeled ssDNA of homologous
sequence is titrated from 0 mMto8mM. UvsX was mixed with
both DNA substrates and a saturating amount of ATP simulta-
neously. Reaction progress curves were fit to an exponential
function to determine the amplitude of fluorescence quenching.
The amplitude was graphed as a function of ssDNA concentration
(Figure 5A). When no ssDNA was present the amplitude reflects
saturated binding of UvsX to dsDNA substrate (compare to
Figure 3. Binding of UvsX to single-standed DNA in the
presence of ATP or ATPcS. Reactions were initiated by the addition
of UvsX (final concentrations indicated) to a mixture of 2 mM
(nucleotides) oligonucleotide dA
22
XA
2
(oligo 1) ssDNA and (A) 2.5 mM
ATP (final concentration) or (B) 900 mM ATPcS (final concentration). The
amplitude of fluorescence quenching was graphed as a function of
UvsX concentrations and fit to Equation 1 to determine an apparent K
d
value.
doi:10.1371/journal.pone.0066654.g003
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 5 June 2013 | Volume 8 | Issue 6 | e66654
Figure 2B). Increasing amounts of ssDNA generated decreased
amplitudes, suggesting less binding of dsDNA (Figure 5A). It could
be argued that the observed changes in Figure 5A are caused by a
simple competition between ssDNA and dsDNA for binding to a
single site in the UvsX filament. However this possibility is ruled
out by the data in Figure 4, which show that dsDNA does not
displace bound homologous ssDNA from the filament. Another
possibility is that the decreased amplitudes are generated as a
result of strand exchange occurring causing the labeled strand to
be displaced. We have also ruled out this possibility by monitoring
DNA strand exchange under identical conditions using a [
32
P]-
labeled oligonucleotide in the place of the AlexaFluor 546–labeled
strand of the duplex (Figure 5B). Reactions were carried out at the
highest concentration of ssDNA used in Figure 5A. As seen on the
gel in Figure 5C, no strand exchange occurs during the time in
which these assays were conducted. Therefore the decrease in
fluorescence observed in Figure 5A is indicative of a decrease in
dsDNA binding and reflects an increased apparent K
d
value for
dsDNA in the presence of homologous ssDNA. The labeled
species added to this reaction was dsDNA however at equilibrium
the labeled species may be part of a strand exchange intermediate
and no longer strictly ‘‘double-stranded’’ (see Figures 6, 7). The
resulting apparent K
d
measurements discussed below, although
strictly discussed as the apparent K
d
for dsDNA in the presence of
homologous ssDNA, may actually be reflective of a 3 strand
intermediate.
To determine the value of this increased apparent K
d
, a titration
of UvsX onto labeled dsDNA in the presence of excess unlabeled
homologous ssDNA was performed (Figure 6). This experiment is
identical to the experiment shown in Figure 2B with the exception
that excess homologous ssDNA is included. Therefore in contrast
to experiments shown in Figure 2B, this experiment measures the
apparent K
d
for dsDNA binding when both the ATP and the
ssDNA binding sites are occupied. Various amounts of UvsX were
added to 2 mM labeled dsDNA (AlexaFluor 546-labeled oligo
1:oligo 2) in the presence of 2.5 mM ATP and 40 mM unlabeled
ssDNA of homologous sequence (oligo 4). UvsX was mixed
simultaneously with both DNA substrates and ATP. The reaction
progress curves were fit to an exponential function to determine
the amplitude of fluorescence quenching, which was then graphed
as a function of UvsX concentration (Figure 6, black circles).
These data were fit to Equation 1 to determine an apparent K
d
of
1.0560.3 mM (solid line). Figure 6 (black circles) shows a binding
curve similar to that of dsDNA binding alone (Figure 2B) in that
the final amplitude of fluorescence quenching is the same.
However more UvsX is required to reach a fully quenched state
indicating that changes in dsDNA binding in the presence of
homologous ssDNA are predominately due to an increase in the
apparent K
d
value for homologous dsDNA binding to a UvsX-
ssDNA complex when compared to binding of dsDNA to UvsX
alone (Table 1). Alternatively, since these reactions in principle
contain the minimum requirements for a strand exchange reaction
to occur (UvsX, Mg
2+
ATP, ssDNA, and homologous dsDNA), the
observed changes in apparent K
d
could reflect a change in affinity
for substrates vs. products. The data in Figure 5C appear to rule
this out, however, since they establish that complete strand
exchange does not occur over a time scale of 0–10 minutes,
whereas all of the binding measurements to determine apparent K
d
values in Table 1 are taken during the first 30 seconds after
mixing. Therefore the data in Figure 6 (black circles) are consistent
with the formation of an intermediate complex containing three
DNA strands bound simultaneously to UvsX (see Discussion and
Figure 7).
Figure 4. Binding of UvsX to ssDNA in the pres ence of
increasing amounts of homologous dsDNA 3 mM UvsX (final
concentration) was added to mixtures containing final con-
centrations of 2 mM ssDNA (dA
22
XA
2
, oligo 1), 2.5 mM ATP,
and various concentrations of homologous dsDNA (dT
25
:dA
25
,
oligo 2:oligo 4). The amplitude of fluorescence quenching was
graphed as a function dsDNA and a line was fit (solid line) to
demonstrate the trend.
doi:10.1371/journal.pone.0066654.g004
Figure 5. Binding of UvsX to dsDNA in the presence of
increasing amounts of homologous ssDNA. A) 2 mM UvsX (final
concentration) was added to a mixture of 2 mM dsDNA (dT
25
:dA
22
XA
2
oligo 2:oligo 1), 2.5 mM ATP, and various amounts of homologous
ssDNA (dA
25,
oligo 4). The amplitude of fluorescence quenching was
graphed as a function of ssDNA concentration and fit to a single
exponential function (solid line) to demonstrate the trend. B) Schematic
of strand exchange reaction used to determine if strand exchange is
occurring during the monitoring of binding reactions depicted in A. C)
Strand exchange reaction containing 0 or 2 mM UvsX, 8 mM ssDNA
(dA
25
) and 2 mM
32
-P labeled dsDNA (dA
25
:dT
25
) in the presence on
2.5 mM ATP. Control lane ‘‘C’’ indicated where on the gel the outgoing
strand would be seen if strand exchange were to occur.
doi:10.1371/journal.pone.0066654.g005
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 6 June 2013 | Volume 8 | Issue 6 | e66654
UvsX Binding to dsDNA in the Presence of ATP and
ssDNA of Heterologous Sequence
Analogous experiments were conducted to investigate the effects
of heterologous ssDNA on the stability of UvsX-dsDNA interac-
tions (Figure 6 gray circles). In these experiments, heterologous
ssDNA (oligo 3) was substituted for homologous ssDNA (oligo 4),
while the labeled dsDNA substrate (AlexaFluor 546-labeled oligo
1:oligo 2) was the same in both data sets depicted in Figure 6. All
other conditions were identical. Differences observed in dsDNA
binding were due to changes in the unlabeled ssDNA present. The
equilibrium data demonstrate a dramatic decrease in binding to
dsDNA in the presence of heterologous ssDNA (Figure 6, gray
circles). The apparent K
d
value for dsDNA under these conditions
was too high to be measured at experimentally attainable protein
concentrations.
Discussion
Results of this study suggest that UvsX recombinase avoids
inhibition by excess non-homologous dsDNA through allosteric
effects mediated by ssDNA binding. In the presence of ssDNA,
dsDNA binding is attenuated and correlated with its homology to
the bound ssDNA. These insights were made possible by a novel
assay that detects the binding of dsDNA to a site on UvsX when
ssDNA occupies a second site, or vice-versa.
AlexFluor 546 as a Probe for UvsX-DNA Interactions
The AlexaFluor 546-DNA conjugate was used as a fluorescence
probe for UvsX-DNA interactions. This probe offers several
advantages including: sufficient brightness for work at nanomolar
concentrations; similar fluorescence signals in ssDNA and dsDNA;
no effect on UvsX-DNA binding affinities. A previous study
employed fluorescein-labeled oligonucleotides as quantitative
probes for UvsX-ssDNA interactions [20]. The AlexaFluor 546-
DNA conjugates provide a similar utility, with the added
advantage of probe brightness, a larger amplitude of fluorescence
quenching by UvsX, and therefore greater sensitivity. Other
studies employed etheno-modified ssDNA containing the fluores-
cent bases ethenoadenine and ethenocytosine [16,30,31]. These
probes provide detailed information on the structural changes
(unstacking, extension) of ssDNA as it is bound by a recombinase.
However ethenobases are known to alter the affinity of
recombinase-ssDNA interactions, and they disrupt base pairing,
preventing their use in dsDNA or in homologous pairing reactions
[16,32]. The AlexaFluor 546 probe avoids this problem and thus
allows direct comparisons of ssDNA and dsDNA binding by
UvsX, and of the effects of one bound lattice on the binding of the
other.
Several observations suggest that general environmental fea-
tures within recombinase-DNA filaments are responsible for the
quenching of AlexaFluor 546 fluorescence, and that signal is
relatively insensitive to filament conformation. Probe fluorescence
is insensitive to the ss/ds character of the DNA it is attached to,
and UvsX binding quenches probe fluorescence to a similar
Figure 6. Binding of UvsX to dsDNA in the presence of excess
homologous or heterologous ssDNA. Various amounts of UvsX
were added to mixtures containing final concentrations of 2 mM dsDNA
(dT
25
:dA
22
XA
2
oligo 2: oligo 1), 2.5 mM ATP, plus 40 mM of either
homologous (dA
25,
oligo 4) or heterologous (dC
25
oligo 3) ssDNA. The
amplitude of fluorescence quenching was graphed as a function of
UvsX concentration. Data for homologous ssDNA were fit to Equation 1
to determine an apparent K
d
value (black line). Data for heterologous
ssDNA were fit to a line (gray line).
doi:10.1371/journal.pone.0066654.g006
Figure 7. Potential structures of the three-stranded intermediate formed in reactions in which homologous substrate sets were
used in the presence of ATP. This figure shows the three intermediates that could form upon mixing a fluorescent dsDNA with homologous
ssDNA and ATP. The three-stranded intermediate formed may be pre-strand exchange, post-strand exchange, or an intermediate. See Discussion for
further information.
doi:10.1371/journal.pone.0066654.g007
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 7 June 2013 | Volume 8 | Issue 6 | e66654
degree on either substrate (Figure 1). Similar amplitudes of
fluorescence quenching are observed when the probe attachment
position on the oligo is changed or when the oligo sequence is
changed (Figures S2, S3). Therefore the quenching of probe
fluorescence appears to be a simple indicator of the occupancy of
DNA by the recombinase. This is supported by the observation
that UvsX quenches the fluorescence of AlexaFluor 546-labeled
oligo dT in the absence or presence of nucleotide (Figure S3),
which rules out the possibility that nucleotide-free UvsX could
somehow occupy ssDNA without quenching the probe.
A minimalist System for the Sensing of Sequence
Homology by UvsX Recombinase
The results of this study provide a framework for understanding
the homologous pairing activity of a recombinase through the
coordination of its ssDNA and dsDNA binding activities. To lower
the complexity of the problem, a minimalist system was devised for
the sensing of sequence homology or heterology by UvsX
recombinase. Homopolymeric DNA substrates were used to avoid
regions of secondary structure in ssDNA, which could bind the
recombinase differentially, and to generate uniformly homologous
or heterologous substrate sets. Short oligonucleotides were used to
simplify DNA binding analyses by minimizing cooperativity.
Binding isotherms in Figures 2–3 are non-sigmoidal, and binding
kinetics have no lag phase (Figure S1 and data not shown),
indicating that UvsX binds non-cooperatively to short oligos. This
is likely due to the fact that UvsX exists in oligomeric structures on
the order of hexamers, which may bind as a unit to short oligos (J.
Liu and S. Morrical, unpublished results). The 25-mers used in our
experiments were designed to accommodate six subunits of UvsX,
equivalent to one hexamer or one helical turn of the presynaptic
filament. Cooperative binding observed on long ssDNA molecules
[16,17] likely involves longer-range interactions between oligo-
meric units of UvsX.
The apparent binding constants reported in Table 1 may be
biased by the homopolymeric sequences and short lengths of these
substrates compared to more physiological DNA substrates.
However the significance of this work lies in the demonstration
of coordination between the ligand binding sites of UvsX (ssDNA,
dsDNA, ATP) rather than in the absolute binding constants for the
model DNA lattices. This coordination is evident from the changes
seen in apparent K
d
values for the same substrate at the same site
as the occupancy of the other sites is changed (Table 1). The
information obtained here using minimalist DNA substrates is
relevant to UvsX interactions with more physiological DNA
substrates. The affinity preference of UvsX for homopolymeric
duplex over ssDNA is consistent with previous findings that: (1)
UvsX binds preferentially to mixed sequence dsDNA over ssDNA
[21]; and (2) The intrinsic affinity parameter (independent of
cooperativity) of UvsX for dsDNA equals or exceeds that for
ssDNA over a wide range of salt concentrations (H. Xu and S.
Morrical, unpublished results). These findings indicate that under
physiological conditions (high dsDNA/ssDNA ratio, relatively
high ionic strength) UvsX filaments would preferentially form on
dsDNA in the absence of other factors.
The minimalist, oligonucleotide-based recombination system
allows for homology detection by UvsX (Figure 6), however
complete strand exchange is not observed (Figure 5C). As depicted
in Figure 7, the three-stranded species detected likely mimics a
homologous pairing intermediate that forms prior to the ejection
of the outgoing strand from the complex. It is significant that the
formation of this species depends on the sequence homology of the
strands present, however our data cannot distinguish between two
possible forms of this complex: the pre- and post-strand exchange
forms (Figure 7). The quenched intermediate may in fact be a
post-strand exchange complex (Figure 7). Complete ejection of the
outgoing strand may not occur under our experimental conditions
due to the absence of accessory proteins such as Gp32, which
stimulates UvsX-catalyzed strand exchanges between long homol-
ogous DNA substrates in part by sequestering the outgoing strand
[33], or UvsW helicase, which stimulates strand transfer by
promoting branch migration [34]. Alternatively, failure to
exchange strands could be due to insufficient filament length or
to effects of the homopolymeric DNA sequences used in our
experiments.
Differential Effects of ATP on UvsX-ssDNA and –dsDNA
Interactions
In the presence of nucleoside triphosphates, recombinases
including UvsX bind and stretch ssDNA to form an active
filament conformation. Recombinase binding and formation of the
stretched conformation is somewhat sequence dependent due to
differential base stacking interactions of the four different bases
[26]. Formation of the stretched conformation is more favorable
within tracts of sequence in which base stacking interactions are
low (e.g. polypyrimidine tracts). In this study we have employed
the fluorescently labeled ssDNA homopolymer dA
22
XA
2
(oligo 1).
This homopolymer is structured and has high base stacking energy
when compared with other sequences, such as poly(dT) [35,36]
and thus the recombinase may bind to it with less affinity due to
the fact that attaining the stretched conformation requires
overcoming more base stacking energy. This could explain our
observation that UvsX binding to dA
22
XA
2
requires ATP or
ATPcS under the conditions of our experiments (Figures 1–3). As
noted previously, binding to oligo dT, a lattice with lower base
stacking energy, is not ATP-dependent (Figure S3). In this respect
oligo 1 is a stringent mimic of ssDNA that is encountered in the
cell.
While ssDNA binding is enhanced by ATP or ATPcS, we
observe that dsDNA binding is destabilized by these nucleotides
(Table 1). Much like ssDNA, dsDNA within the ATP containing
filament may be stretched. Previously, the low-resolution struc-
tures of UvsX-dsDNA filaments were solved in the presence of
ADP-AlF
4
or of ADP by cryo-electron microscopy [18]. The ADP-
AlF
4
structure was stretched by 150% compared to the structure in
the presence of ADP. Recently an X-ray crystal structure of a
truncated form of UvsX was solved [34]. This atomic structure
was modeled into the UvsX filament structures solved by cryo-
electron microscopy. The investigators report that a nucleotide
cofactor cannot be accommodated in the condensed filament [34].
These findings suggest that the low-K
d
form of UvsX-dsDNA that
we observe in the absence of ATP represents a condensed
filament, while the high- K
d
form seen in the presence of ATP
represents a stretched filament.
Mobilization of UvsX from dsDNA in Respons e to ssDNA
The binding of UvsX to ssDNA is necessary to activate its
catalytic activities. The vast majority of DNA in the T4-infected
cell is double-stranded, and has the potential to be a potent
inhibitor of UvsX facilitated recombination. In fact UvsX binds
with greater affinity to dsDNA alone than to ssDNA alone
(Table 1; [21] and unpublished results). Binding to dsDNA does
not activate the ATPase activity of UvsX [22] (data not shown).
This lack of ATP hydrolysis is not due to a lack of ATP binding,
since binding to dsDNA is weakened in the presence of ATP
(Table 1). The apparent K
d
of the UvsX-dsDNA complex is still
relatively low (ca. 200 nM) in the presence of ATP however,
suggesting that the enzyme seldom exists in the DNA-free form.
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 8 June 2013 | Volume 8 | Issue 6 | e66654
We propose instead that UvsX-dsDNA complexes represent a
reservoir or sequestered form of inactive recombinase, which
avoids the nonproductive consumption of ATP until recombina-
genic ssDNA is generated (Figure 8).
The data in Table 1 indicate that ATP-induced affinity changes
are insufficient to favor a simple exchange of UvsX from dsDNA
to ssDNA. Instead, pre-formed UvsX-dsDNA complexes appear
to be actively remodeled by ssDNA. The presence of heterologous
ssDNA dramatically destabilizes UvsX-dsDNA binding, while the
presence of homologous ssDNA raises the apparent K
d
for dsDNA
by approximately 5-fold (Figure 6, Table 1). It is clear from the
data that the homologous ssDNA and dsDNA molecules do not
compete for binding to a single site within the UvsX filament
(Figures 4 and 5). Instead the data favor a model in which ssDNA
binding allosterically alters the apparent K
d
for dsDNA binding to
a second site within the UvsX filament. Together, the binding data
are consistent with a model in which UvsX is tightly associated
with dsDNA when recombination is not occurring in the cell
(Figure 8). When present on dsDNA, UvsX can bind but not
hydrolyze ATP. ssDNA generated as a result of DNA damage
binds to the duplex-bound UvsX, activating it for recombination
and diminishing its affinity for heterologous dsDNA. Thus, if
duplex-bound UvsX can be thought of as a reservoir or
sequestered form of inactive recombinase, the generation of
ssDNA mobilizes the enzyme, releasing UvsX from unproductive
association with random dsDNA while ‘‘tuning’’ the enzyme to
discriminate between dsDNA sequences on the basis of homology
to the ssDNA present (Figure 8). The transfer of UvsX from
dsDNA to ssDNA is necessary to activate ATP hydrolysis and
exchange, which in turn facilitates conformations of the filament
that promote D-loop formation upon recognition of homologous
DNA.
Accessory Proteins may also Mitigate Potential Inhibitory
Effects of dsDNA
While UvsX itself appears to have the ability to clear itself from
potentially inhibitory dsDNA in response to a ssDNA signal, other
proteins are also needed to form and/or stabilize an active
presynaptic filament, and these proteins may also help to confer to
UvsX a selective affinity for ssDNA. UvsY, a recombination
mediator protein required for UvsX recombination transactions
in vivo, has a strong bias toward binding to ssDNA and may
promote the selective nucleation and stabilization of UvsX
filaments on ssDNA [9,20,37]. UvsX and UvsY work in concert
with Gp32, which plays an important role in presynaptic filament
assembly by denaturing inhibitory secondary structure in ssDNA
before being displaced by UvsX/UvsY [11,38]. These two
accessory proteins together with the intrinsic coordination of the
two DNA binding sites of UvsX demonstrated here, may help
stabilize active UvsX filaments on ssDNA once it has been
mobilized from association with random dsDNA in an unproduc-
tive/sequestered complex.
In vitro DNA strand exchange assays with many recombinases
are typically staged in such a way that the recombinase filament is
pre-assembled on ssDNA before dsDNA is introduced into the
reaction. Physiologically, recombinases almost always encounter
substrates in the opposite order–dsDNA before ssDNA. Eukaryotic
organisms appear to rely on the DNA translocase activity of
Rad54 to remove Rad51 recombinase from random dsDNA as a
prerequisite to presynaptic filament assembly on ssDNA [39].
Rad51-dsDNA complexes turn over very slowly in the absence of
Rad54, leading to inhibition of strand exchange due to excess
duplex [40]. In the T4 recombination system, UvsX-dsDNA
complexes appear to turn over efficiently in response to ssDNA,
circumventing the need for a Rad54-like DNA translocase activity.
T4 relies on homologous recombination events to initiate genomic
replication during late stages of infection in E. coli cells, resulting
in a greatly amplified phage burst [11,41]. The enhanced
dynamics of UvsX-DNA interactions allows the phage to rapidly
Figure 8. Model for ssDNA recognition and activation of recombination in the presence of excess dsDNA. (A) In the presence of only
dsDNA, UvsX has high affinity for dsDNA and low turnover of ATP. (B) In the presence of both ssDNA and dsDNA UvsX has higher affinity for ssDNA
(C) and affinity for dsDNA is directly correlated to its homology to the ssDNA bound by the enzyme. (D) The ssDNA bound by the enzyme dictates the
search for homology within the dsDNA. See text for details.
doi:10.1371/journal.pone.0066654.g008
Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 9 June 2013 | Volume 8 | Issue 6 | e66654
mobilize its recombination machinery to facilitate recombination-
dependent replication.
Supporting Information
Figure S1 Typical traces of fluorescence quenching data
used to measure dissociation constants for UvsX and
labeled DNAs in the presence of nucleotide cofactors.
UvsX hydrolyzes ATP in the presence of ssDNA. ATP hydrolysis
is associated with the release of ssDNA. Thus the Uvs-
X:ATP:ssDNA tripartite species is transient. In order to measure
the affinity of UvsX for ssDNA in the presence of ATP rapid
mixing techniques were used to observe ssDNA binding before
ATP hydrolysis. We also used ATPcS, an ATP analogue which is
hydrolyzed slowly. Reactions were initiated by the addition of
UvsX (final concentrations indicated) to a mixture of 2 mM
(nucleotides) ssDNA and (A) 2.5 mM ATP (final concentration) or
(B) 900 mM ATPcS (final concentration). Reaction progress was
monitored for up to 1000 s however only the first 30 s were used
to determine the binding constants (Table 1). These data were fit
to an exponential function to determine the total amplitude of
quenching at each protein concentration. These amplitudes were
then plotted as a function of UvsX concentration and these data
were fit to Equation 1 to determine an apparent K
d
. The similarity
of the ATP and ATPcS fluorescence data as well as the similarity
of the apparent K
d
values obtained lead us to conclude that our
techniques allowed us to measure the ATP bound form of the
enzyme in the presence of ssDNA.
(TIF)
Figure S2 Effects of UvsX protein on fluorescence
emission spectra of AlexaFluor 546-labeled oligonucleo-
tide with alexafluor 546 differentially positioned. A
single-stranded 25 mer oligonucleotide of the sequence dA
25
with
a59 C6 amino-modifier was covalenty labeled with AlexaFluor
546. The fluorescence emission from 560–600 nm with an
excitation of 554 nm was recorded for 2 mM of this oligonucle-
otide alone (solid line), after the addition of 1.35 mM UvsX
(dashed line) and after the addition of 3 mM ATP (dotted line).
Reaction conditions were as described in the material and
methods section for steady-state measurements.
(TIF)
Figure S3 UvsX binding to 25 mer oliognucleotide 59-
dT
22
XT
2
-39, in which X is amino-modifier C2 dT where
the alexa 546 probe is covalently attached. 2uM ssDNA
was added to various amounts of UvsX in the presence and
absence of 900 mM ATPcS. The (A) fluorescence quenching
relative to ssDNA alone and the (B) amplitude of fluorescence
quenching was graphed as a function of UvsX concentration.
(TIF)
Figure S4 Titration of nucleotide cofactors to determine
saturated binding conditions. A nucleotide cofactor is
required for UvsX to binding to single-stranded oligo 1. ATP
and ATPcS titrations were conducted to determine a saturating
amount of ATP and ATPcS to be used in the ssDNA binding
assays. Rapid mixing techniques with a SX.18 MV stopped-flow
fluorometer (Applied Photophysics, Leatherhead, Surrey, UK)
were used to measure binding of UvsX to ssDNA in the presence
of the nucleotide cofactor before hydrolysis. 2 mM AlexaFluor 546
labeled oligo 1 and 1.35 mM UvsX were rapidly mixed with 0–
3 mM ATP or ATPcS in a reaction buffer containing 20 mM
Tris-HCl, pH 7.4, 50 mM NaCl, 3 mM MgCl
2
. The amplitude of
fluorescence quenching was graphed as a function of ATP or
ATPcS concentration. From these data 900 mM ATP cS and
2.5 mM ATP were chosen as saturating concentrations used to
measure DNA binding by UvsX.
(TIF)
Figure S5 Stoichimetric binding of UvsX to dsDNA.
Various amounts of UVSX was added to 2 mM (nucleotide pairs)
dsDNA in the absence of a nucleotide cofactor. Three separate
experiments were conducted as specified in the material and
methods section. Total change in fluorescence was graphed as a
function of UvsX concentration. Data were fit to Equation 1 (solid
line). The binding site size indicated by these data is 4 nucleotide
pairs/UvsX monomer (2 mM nucleotide pairs is saturated by
0.5 mM UvsX).
(TIF)
Acknowledgments
DNA sequencing and phosphorimaging facilities were provided by the
Vermont Cancer Center.
Author Contributions
Conceived and designed the experiments: RLM SWM. Performed the
experiments: RLM. Analyzed the data: RLM. Contributed reagents/
materials/analysis tools: RLM. Wrote the paper: RLM SWM.
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Coordinated ss/dsDNA Binding by UvsX Recombinase
PLOS ONE | www.plosone.org 11 June 2013 | Volume 8 | Issue 6 | e66654
    • "Alternatively, recovery from dsDNA may involve ligand-induced allosteric effects on the DNA-pairing protein itself. This is observed with the T4 UvsX protein, in which ATPase-inactive complexes on dsDNA are rapidly activated for DNA strand exchange by the addition of homologous , but not of heterologous, ssDNA (Maher and Morrical 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways-DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
    Article · Feb 2015
    • "The primers used were below the minimum length required (determined here to be approximately 25–30 nucleotides) for the formation of a recombinase/oligonucleotide complex (pre-synaptic filament), which is necessary for the insertion of a single-stranded DNA molecule into a target duplex. The IO used in SIBA is above this length, enabling its recombinase-dependent insertion into the target duplex [8,18,20]. These criteria were confirmed under the SIBA reaction conditions (Figs S1 and S2). "
    [Show abstract] [Hide abstract] ABSTRACT: Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.
    Full-text · Article · Nov 2014
  • [Show abstract] [Hide abstract] ABSTRACT: Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX–ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.
    Article · Oct 2013
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